DE69017324T2 - Living tissue equivalent. - Google Patents

Abstract

The present invention provides improved tissue equivalents and methods of making and using such tissue equivalents. The present invention also provides test apparatus incorporating such improved tissue equivalents.

Description

FIELD OF INVENTION

This invention relates to methods of making and using tissue equivalents comprising (i) a hydrated collagen lattice contracted by a contractile agent and (ii) a collagen gel in contact with a permeable element. This invention further relates to assay systems containing such tissue equivalents.

Tissue equivalents containing a hydrated collagen lattice that is contracted by a contractile agent such as fibroblast cells or platelets to form the tissue equivalent are shown in U.S. Patent Nos. 4,485,096; 4,485,097; 4,539,716; 4,546,500; 4,604,346; and 4,837,379 and in copending U.S. Patent Application Serial No. 07/252,249 filed September 30, 1988 (EP-A-89309972.1), which are incorporated herein by reference in their entirety (hereinafter collectively referred to as "the patents"). Tissue as used herein includes any group or layer of cells that together perform one or more specific functions. Such tissue equivalents include, but are not limited to, Equivalents of epithelial tissue, connective tissue, cartilage, bone, organs, glands and blood vessels and include living cells and extracellular matrix molecules, primarily collagen, and may optionally contain components not typically found in normal tissues.

Generally, such living tissue equivalents are prepared by forming a hydrated collagen lattice that is contracted into a living tissue. Examples of contractile agents include fibroblast cells, smooth muscle cells, and platelets. A skin equivalent can be prepared from the living connective tissue equivalent substrate by plating keratinocyte cells thereon and allowing them to grow.

The tissue equivalents referred to above are populated with cells that can remain alive for long periods of time and can be produced on a larger scale while ensuring that the units produced are substantially uniform. Cells in such tissue equivalents resemble cells of normal tissue in their structural arrangement, biosynthetic capacity and permeability. It is understood that these tissue equivalents need not be of human origin but can be of any animal if desired.

Tissue equivalents in accordance with the teachings of the present invention have a wide range of applications including applications in research and development, tissue and organ replacement, and testing.

Human skin tissue equivalents allow the growth of normal human epidermal cells that are fully differentiated, which have not previously been obtained by routine culture methods. Such skin tissue equivalents have been widely used as permanent skin substitutes in animal experiments The morphological appearance of such skin tissue equivalents is normal, the cells that make up the components persist after transplantation as demonstrated by genetic marking, and functional capacity has been demonstrated. See, e.g., Science, 211: 1052-1054 (1981); J. Invest. Dermatol. 81: 2s-10s (1983).

Skin tissue equivalents prepared in vitro by the methods shown in the patents closely resemble natural skin. Such equivalents comprise a multilayered epidermis with well-developed basal cells connected to form a dermal layer. The dermal layer comprises a collagen matrix in which dermal fibroblasts are distributed. Cells in the three-dimensional collagen matrix achieve a state of differentiation that is in many respects similar to that which exists in vivo. For example, resident fibroblasts are synthetically active and enrich the matrix in vitro with both collagen and a number of other molecular species and exhibit permeability properties typical of a cell in vivo. See, e.g., Collagen Rel. Res. 4: 351-364 (1984). Furthermore, skin tissue equivalents can be pigmented by inclusion of melanocytes, which deliver pigment to keratinocytes, and the process has been shown to be accelerated in vitro by ultraviolet radiation (J. Invest. Dermatol. 87: 642-647, 1986).

US Patent No. 4,835,102 discloses systems containing tissue equivalents that reproduce in vitro many of the physical and biological properties of natural tissue and are useful for studying, for example, tissue cell biology, physiology and pathology. Such systems include devices and kits for determining the interaction of tissue and at least one agent using at least one tissue equivalent. The agent includes, but are not limited to various substances such as chemicals, cosmetics, pharmaceuticals; stimulants, e.g. light or physical injury; and tissue protecting agents.

Tissue equivalents can be cast in any desired shape. Skin tissue equivalents for use in skin grafts and in test systems are generally cast as flat slabs, and blood vessel equivalents are generally manufactured as a hollow tube or as a network of hollow tubes. The natural geometry or configuration of the tissue equivalent can, however, be altered as desired. For example, a skin tissue equivalent can be cast as a cylinder rather than a slab.

When fibroblasts or smooth muscle cells are used as the contractile agent in the preparation of tissue equivalents, an unrestrained collagen lattice typically undergoes contraction in all dimensions. Various methods to assist in the formation of living tissue and tissue equivalents with controllable configurations and/or dimensions are shown in the patents, e.g., U.S. Patent No. 4,485,096. For example, a collagen lattice can be cast on a stainless steel frame to keep the boundaries fixed and thus perform contraction essentially only in the thickness direction. Other materials that can be used to control the configuration of the fabric equivalents include VELCRO, textured plastics, and textured TEFLON. Although these restraints are effective, in many cases it is preferable, both in terms of cost and simplicity, to use restraints that are not integral with the mold chamber.

Considering the diverse applications of tissue equivalents Improved tissue equivalents and processes for producing such tissue equivalents are sought.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a partially broken away isometric view of an apparatus according to the present invention.

Figure 2 is an exploded isometric view of the device shown in Figure 1.

Figure 3 is a sectional view taken along 3-3 through the device shown in Figure 1.

Figure 4 is a partially broken away isometric view of another device according to the present invention.

Figure 5 is an exploded top view of the device shown in Figure 4.

Figure 6 is an exploded sectional view taken along 6-6 through the device shown in Figure 4.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides tissue equivalents comprising (i) a hydrated collagen lattice contracted by a contractile agent and (ii) a collagen gel. The present invention further provides tissue equivalents having improved properties including increased sample-to-sample reproducibility in the degree of contraction and, in the case of skin tissue equivalents, in the degree of differentiation of the epidermis.

In some embodiments of the present invention, such fabric equivalents adjacent to one or more absorbent elements including fibrous pads and gels such as agarose.

The present invention further provides methods for making and using such improved tissue equivalents. The methods taught herein also offer advantages over those methods shown in the patents in terms of ease of manufacture and reduced manufacturing costs.

A method according to the present invention by which a tissue equivalent is formed comprises:

(a) forming a mixture comprising collagen and at least one contractile agent; and

(b) applying the mixture obtained in step (a) to a collagen gel and maintaining the mixture and the gel under conditions that allow the formation of the tissue equivalent.

The present invention further provides a device containing such tissue equivalents for determining the interaction of tissue and one or more agents.

In a preferred embodiment of the present invention, such a device comprises a first and a second region formed by a permeable device provided with a collagen gel on which a tissue equivalent is disposed, the tissue equivalent defining the first region and the permeable device defining the second region.

In a further preferred embodiment, such a device comprises at least one tissue equivalent and at least one container for the tissue equivalent, wherein the Container includes:

(i) an inner container comprising a permeable device provided with a collagen gel on which the tissue equivalent is disposed, the permeable device forming the bottom of the inner container, and

(ii) an outer container within which the inner container is arranged.

DETAILED DESCRIPTION OF THE INVENTION

The tissue equivalents of the present invention, while similar in both methods of manufacture and use to those containing a hydrated collagen lattice as shown in the patents, further comprise a layer of collagen.

It has been unexpectedly found that a collagen lattice cast on a collagen gel in contact with a permeable element does not undergo significant radial or lateral contractions as it contracts in the thickness direction, thereby eliminating the need to anchor the collagen lattice to, for example, a stainless steel frame to control radial or lateral contraction. For example, a 24 mm diameter collagen lattice cast according to the teachings of the patents, but without an anchoring device, would contract radially to a diameter of 5 mm or less. In contrast, a 24 mm diameter collagen lattice cast on a collagen gel in contact with a permeable element typically contracts radially to a diameter of about 15 mm. Elimination of the anchoring device to control lateral/radial contraction offers advantages in terms of cost reduction and simplification of fabrication of tissue equivalents.

It will be understood that such an anchoring device can be used in conjunction with a collagen gel in contact with a permeable element if desired. A method for obtaining the tissue equivalents according to the present invention comprises:

(a) forming a mixture comprising collagen and at least one contractile agent; and

(b) applying the mixture obtained in step (a) to a collagen gel in contact with a permeable element and maintaining the mixture and the gel under conditions allowing the formation of the tissue equivalent.

In some embodiments of the present invention, one or more absorbent elements, including, but not limited to, fiber pads, cotton pads, and gels, agarose, are used in conjunction with the collagen gel described above. Such absorbent elements have been found to provide a consistent and even physical support and promote uniform contact between the tissue equivalent and the cell culture medium. Typically, the absorbent element is adjacent to the surface of the collagen gel opposite the hydrated collagen lattice. If the absorbent element or elements themselves are a gel, e.g., agarose, the gel may be provided with a nutrient medium to provide nutrients to the tissue equivalent.

The following experimental endpoints were monitored in tissue equivalents maintained with and without collagen gel and/or the absorbent element during different stages of development of a skin tissue equivalent:

1. Glucose utilization;

2. Extent and quality of epidermal stratification and keratinization;

3. pH of the medium.

The observed pH values of media obtained from tissue equivalents maintained with the absorbent element(s) were consistently higher and closer to physiological pH than tissue equivalents maintained in the absence of these elements. In addition, glucose utilization was observed to be generally lower in tissue equivalents maintained with the absorbent element.

Surprisingly, it was found that mature keratinization is promoted in skin tissue equivalents prepared using the absorbent element(s) as compared to control skin equivalents prepared without such elements. Although the mechanism by which these absorbent elements may affect epidermal differentiation is unknown, it is postulated that such elements may act as diffusion barriers, e.g., a permeability barrier, and may filter the medium and/or retain secreted cellular products in close proximity to the tissue equivalents.

Living skin equivalents according to the present invention are prepared as described in the patents, with the exception that in accordance with the present invention, the hydrated collagen lattice is cast onto a collagen gel.

A method of making a skin tissue equivalent in accordance with the present invention comprises:

(a) forming a mixture comprising collagen and at least one contractile agent;

(b) applying the mixture obtained in step (a) to a collagen gel in contact with a permeable element and maintaining the mixture and the gel under conditions that allow the formation of the tissue equivalent; and

(c) seeding the tissue equivalent obtained in step (b) with keratinocytes.

By way of background information, a common protocol for casting the tissue equivalents of the present invention involves rapidly mixing an acidic collagen solution having a pH of about 3 to 4 with nutrient media, adjusting the pH of the resulting solution, if necessary, to about pH 6.6 to pH 7.8, adding fibroblast cells, transferring the resulting mixture ("the casting mixture") to a suitable mold or casting device in which a collagen gel is disposed, and then incubating at a temperature of about 35°C to 38°C. A most convenient approach is to adjust the pH while simultaneously combining the ingredients of the casting mixture. These steps can be performed in any desired order, provided that the steps are completed so that the casting mixture can be transferred to a mold for proper curing. The collagen fibrils are precipitated from the casting mixture as a result of heating the solution and increasing the pH to form a hydrated collagen gel that is contracted by a contractile agent and arranged on a collagen gel.

Although the methods for producing living tissue provided by the present invention are applicable to the production of tissue equivalents in general, these methods are used in connection with the production of skin equivalents for use in skin transplantation applications and in test systems containing skin equivalents.

Devices, methods and kits for determining the interaction of tissues and one or more agents using tissue equivalents, including skin tissue equivalents, are described in U.S. Patent No. 4,835,102. Tissue equivalents for such applications may also advantageously further comprise, in accordance with the present invention, a collagen gel in contact with a permeable element.

In the figures, Figures 1-3 show an embodiment of an apparatus for determining the interaction of skin and one or more agents using skin tissue equivalents in accordance with the present invention, wherein multiple containers 10, 22 in which tissue equivalents according to the present invention are arranged are arranged in a base or holder. The device shown in Figures 1-3 is further provided with a covering device 2. In some embodiments, a cover (not shown) is provided for each container 10, 20. The cover is selected from any biocompatible material that seals the container. Suitable cover materials include foils and barrier films that can be sealed to the device by means of an adhesive or by means of heat. A heat-sealable polyester film is particularly useful in the practice of the present invention.

The containers for the tissue equivalents comprise an outer container 10 and an inner container 20. The inner container 20 is provided with a rim 50 to form a means for positioning the inner container 20 within the outer container 10, thereby defining an outer region 14 and an inner region 22. The inner container 20 is provided with a skin tissue equivalent 26, 28 disposed on a collagen gel 25, which in turn is disposed immediately adjacent a permeable element 24. The permeable element is sealingly attached to the inner container 20 and forms the bottom surface thereof. The skin tissue equivalent comprises two layers 26, 28, layer 28 comprising an epidermal layer and layer 26 comprising a dermal layer. In some embodiments, a sealing element 30 creates a seal between the inner wall of the inner container 20 and the skin equivalent 26, 28 and covers the periphery of the collagen gel 25 in the case where the outer edge of the tissue equivalent 26, 28 is disposed within the collagen gel 25. In the embodiments depicted in the figures, the container 10 is provided with an opening 21 providing access to the outer region 14.

The device shown in Figure 3 is further provided with an absorbent element 32. In further embodiments, a gel (not shown) can be arranged in the outer chamber 14.

Figures 4-6 illustrate another embodiment of a device for determining the interaction of tissue and one or more agents using tissue equivalents in accordance with the present invention. Elements similar to those in other described embodiments are designated by the same reference numeral. In this embodiment, the outer container 10 is rectangular and provided with raised portions 60 in the bottom on which the inner container 20 can be placed. The outer container 10 does not protrude upward from the bottom of the device, but rather is formed as a pocket from the top of the device.

The outer container 10 is provided with nutrient medium for the tissue equivalent. Such media are, according to the state of the art, The volume of the medium must be selected to fill the outer container 14 to an appropriate level so as not to build up a column of pressure which would force the medium through the permeable element 24, the collagen gel 25, the tissue equivalent 26, 28 into the inner container 20.

In some embodiments of the present invention, the outer container 10 is provided with an absorbent element 23 that is disposed within the outer container 10 so as to be in contact with the outer surface of the permeable element 24. The absorbent element 23 must be compatible with living tissue equivalents. Preferred materials for the absorbent element include cotton, polyester and rayon. A particularly preferred material is absorbent cotton. Generally, it is preferred that the absorbent element be free of additives such as detergents.

In other embodiments of the present invention, the outer container 10 is provided with a gel (not shown), such as agarose, which serves to enclose the media. Since the agarose gel can be added to just below the level of the openings 24, a greater amount of nutrient media is made available to the tissue equivalent and the nutritional supply for the tissue equivalent 26, 28 is not as quickly depleted. The use of such gels provides advantages in the storage and shipping of the device of the present invention in terms of minimizing leakage of the media and the resulting possible contamination of the tissue equivalent.

The outer container 10 and the inner container 20 may be made of any desired material that does not reacts with or has an undesirable effect on the test components, including the tissue equivalent. For example, the casting mixture for the living tissue equivalent must not adhere to the walls of the inner container during contraction so as to interfere with the formation of the tissue equivalent.

It has been found that methods used to sterilize the inner container 20 can affect the adhesion of the tissue equivalent. For example, the forming tissue adheres to polystyrene, a preferred material for the inner container, when it is sterilized by electron beam, but not when it is sterilized with ethylene oxide. In contrast, K-resin, an alloy of polystyrene and butadiene and a particularly preferred material for the container, can be sterilized by electron beam without causing adhesion of the forming tissue (K-resin is a trademark of Phillips Petroleum). In some embodiments, it is desirable that the containers be manufactured so that the tissue equivalent is visible through the container, e.g., through the walls of the container or through a window in the container. Preferred materials for the container 10 include polystyrene and PETG.

The inner container 20 can be made in any shape and volume that accommodates the size and shape of the desired tissue equivalent. The dimensions of the container, in turn, are dependent on the size and shape of the desired tissue equivalent and the desired volumes of study. For example, a container with an outer diameter of about 25 mm and a volume of about 5 ml is useful in the practice of the present invention. In the embodiment shown in Figures 1-3, multiple containers are provided in a base or holder.

The permeable element 24 must have sufficient strength to support the collagen gel 25 and tissue equivalent 26, 28. Porous membranes are useful in the practice of the present invention. The pore size of such membranes is selected to provide an attachment for the collagen gel 25. Preferred membranes are hydrophilic, have a thickness of about 1 to 10 mm and pore diameters in the range of about 1 to 10µ. Preferred materials for the permeable element 24 include polycarbonate. A particularly preferred permeable element is a polycarbonate membrane, preferably free of surfactants, which is commercially available from Nuclepore and has a pore size of about 3 to 10µ.

The sealing element 30 can be made of any material that is inert to the experimental conditions and tissue equivalents used and provides a good seal between the inner container 20 and the tissue equivalent. Preferred materials include polyethylene, TEFLON, polycarbonate and nylon. The sealing element is particularly useful when it is desired to keep the contents of the outer container separate from any solution or substance applied to the epidermis 25, for example when measuring the diffusion or permeation of a substance through a tissue equivalent.

Both the tissue equivalents and the collagen gel in accordance with the present invention can be prepared using collagen obtained from skin and tendons, including rat tail tendons, calf skin collagen and calf extensor tendon. Other sources of collagen would be suitable. A particularly preferred collagen composition obtained from the common digital extensor tendon of the calf and methods for obtaining such collagen compositions are set forth in co-pending U.S. Patent Application Serial No. 07/407,465, which is filed with the same application. Date this application was filed and the disclosure of which is incorporated herein by reference.

In a method according to the present invention, the collagen gel 25 is prepared from a collagen composition containing collagen at about 0.5 to 2.0 mg/ml, preferably about 0.9 to 1.1 mg/ml, and nutrient media. This collagen composition is added to the inner container 20 and maintained under conditions that allow the collagen composition to harden and form a collagen gel of suitable dimensions, typically about 1 to 5 mm thick, with a preferred thickness range being about 2 to about 3 mm. The collagen gel 25 is preferably thick enough that a portion remains cell-free when cells migrate from the tissue equivalent into the collagen gel and thin enough that the tissue equivalent is not undesirably removed from the nutrient source provided in the outer container 10.

A dermal equivalent is subsequently cast onto the collagen gel using procedures consistent with the patents and as described below. A casting mixture containing collagen and fibroblasts is added to the inner container 20 over the collagen gel 25 and maintained under conditions that allow the formation of the tissue equivalent. As the tissue equivalent forms on the collagen gel 25, it contracts radially. However, the collagen gel 25 prevents excessive radial contraction of the tissue equivalent without the need for mechanical restraints such as textured metals and plastics or VELCRO.

Typically, the sides of the tissue equivalent 26 are inclined towards the outer periphery of the collagen gel 25 and form a plateau, as shown in Figures 3 and 6 at 52. The tissue equivalent 26 is now seeded with epithelial cells to form the epidermal to form layer 28. The epidermal cells are seeded into the culture medium at a concentration of about 0.3 x 10⁶ to 30 x 10⁶ cells/ml. The volume of epidermal cells seeded depends on the size of the plateau.

The concentration of collagen, the number of cells and the volume of the casting mixture can be controlled to optimize the diameter and thickness of the living tissue equivalent. The casting mixture comprises cells at a concentration of about 1.25 to 5 x 104 cells/ml and collagen at about 0.5 to 2.0 mg/ml in a nutrient medium. A preferred cell concentration is about 2.5 x 104 cells/ml. The ratio of the volume of the casting mixture for the tissue equivalent to the volume of the casting mixture for the collagen gel has been found to have an effect on the viability and differentiation of the cells. Useful ratios v/v of tissue equivalent casting mixture to collagen gel casting mixture are about 3:1 to 1:3. A preferred ratio at which the cell concentration in the collagen lattice is about 2.5 x 10⁴ cells/ml is 3:1.

The invention will be better understood by reference to the following examples, which are purely exemplary in nature and are not to be used as limiting the scope of the invention.

The materials used in the following examples were obtained from the sources indicated in the examples or prepared in accordance with the publications indicated. Sterile procedures were used in all examples. The tissue equivalents were maintained under 10% CO2 in the incubator and sterile procedures were used throughout.

Example I: Production of skin tissue equivalent test systems

A. A device similar to that shown in Figure 4 was used to perform the tasks described below. The cover is removed to perform the task but is otherwise kept in place to maintain sterility. Relevant information regarding the device is listed below:

outer container 10: diameter 38 mm Capacity 35 ml

inner container 20: diameter 24 mm Capacity 4 ml

Permeable element 24: polycarbonate membrane from Nuclepore, pore size 3u, thickness 5u.

B. A collagen gel was formed on the permeable element 24 as follows:

(1) Premix:

10X MEM 16.2ml

L-Glutamine (200 mM) 1.6 ml

Gentamicin (50 mg/ml) 0.2 ml

fetal bovine serum 18.0 ml

Sodium bicarbonate (71.2 mg/ml) 5.0 ml

The batch solutions were mixed at 37ºC and combined in the order above and stored at 4ºC for approximately 30 minutes in 50 mL tubes (not gas treated).

(2) 27.8 g of a 1 mg/ml collagen solution (extracted by acid from the common digital extensor tendon of the calf) in 0.05% v/v acetic acid was weighed into a 50 ml tube and stored at 4ºC for 30 minutes.

(3) 8.2 ml of the premix described above and 4 ml of DMEM complete solution (containing 10% FBS, 4 mM L-glutamine, 50 ug/ml gentamicin) were added (and 1 ml aliquots were pipetted into the inner container 20 and allowed to gel in a lid).

C. Tissue equivalents were cast with human dermal fibroblasts and seeded with human epidermal (epithelial) cells as described below.

A general description of the procedures and reagents can also be found in the patents and copending application, serial number 07/361,041, filed June 5, 1989 (EP-A-90306084.6).

(1) Casting mixture:

8.2 ml of the premix described above was added to 27.8 g of a 1 mg/ml collagen solution in 0.05% v/v acetic acid as described in step A (2) above and 4 ml of human dermal fibroblasts (2.5 x 105 cells/ml) were combined. 3 ml aliquots were pipetted into the container 20 over the collagen gel formed in step B (2) above and allowed to gel. 4.5 ml of DMEM whole solution was added to the outer container 20 and subsequently incubated at 36°C/10% CO2 for typically 4-8 days.

The following medium was used: Component Hydrocortisone Insulin Transferrin Triiodothyronine Ethanolamine O-Phosphorylethanolamine Adenine Progesterone Selenium Bovine Serum Epidermal Growth Factor Calcium-free DMEM Ham's

Two other media used were identical to mSBM, with the following exceptions: cSBM Main SBM Progesterone Bovine Serum Epidermal Growth Factor Calcium-free DMEM Ham's

In some cases calcium chloride was added to the media at 1.8 mM. This is indicated as medium plus calcium, e.g. mSBM plus calcium.

D. Epidermalization was initiated 6 days after casting the tissue equivalent.

(1) Epidermalization

The medium from step C above was removed from both the inner container 20 and the outer container 10. A 50 µl suspension of human epidermal cells (3.33 x 10⁶ cells/ml) was placed on the tissue equivalent formed in step C above. The container was then incubated at 36°C and 10% CO₂ for 4 hours. 12.0 ml of mSBM was then added to the outer chamber and 4 ml to the container. The device was then returned to the same incubator.

(2) Differentiation

52 days after epidermalization, the medium was removed and replaced with mSBM plus calcium.

(3) Airlifting

5 days after epidermalization, the following procedure was performed:

The medium was removed from the inner and outer chambers. The inner container 20 was removed and cotton pads soaked with 2 volumes of cSBM plus calcium were placed on the bottom of the outer chamber 10 and 9.0 ml of cSBM plus calcium was added. The inner container 20 was then replaced and the device incubated at 35.5°C and 10% CO2.

(4) Entertainment

Every 4 days the medium was removed and replaced with fresh main SBM plus calcium.

Example II: Preparation of agarose-containing test systems

A 2% agarose solution in water was sterilized by autoclaving, cooled to 40ºC and mixed with an equal volume of 2% concentrated main SBM. The absorbent cotton pads were removed and 13 ml of the mixture was poured into the outer region 14, curing was allowed at 368C (10% CO2) and the device was returned to the incubator for storage prior to shipping.

It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in view thereof which will occur to one of ordinary skill in the art are intended to be included within the spirit and scope of this application and the scope of the affirmed claims.

Claims (19)

1. Tissue equivalent comprising: (a) a hydrated collagen lattice contracted by a contractile agent; and (b) a collagen gel in contact with a permeable element. 2. A process for producing a tissue equivalent, which process comprises: (a) forming a mixture comprising collagen and at least one contractile agent; and (b) applying the mixture obtained in step (a) to a collagen gel in contact with a permeable element and maintaining the mixture and the gel under conditions that allow the formation of the tissue equivalent. 3. The method of claim 2, wherein the contractile agent comprises fibroblast cells. 4. The method of claim 2, wherein the mixture of step (a) contains fibroblast cells at a concentration of about 1.25 to 4 x 10⁴ cells/ml and collagen at about 0.5 to 2.0 mg/ml. 5. The method of claim 2, wherein the collagen gel is prepared by casting a collagen solution containing collagen at a concentration of about 0.5 to 2.0 mg/ml. 6. The method of claim 5, wherein the mixture of step (a) and the solution of step (b) are used in a ratio of about 3:1 to 1:3, volume:volume. 7. The method of claim 2, wherein an absorbent element is disposed immediately adjacent to the permeable element. 8. The method of claim 7, wherein the absorbent element comprises a fibrous pad or a gel. 9. The method of claim 8, wherein the fiber pad is cotton. 10. The method of claim 2, wherein an agarose-containing gel is disposed adjacent to the permeable element. 11. Device for determining the interaction of tissue and at least one agent using at least one tissue equivalent, which device comprises a first and a second region formed by a permeable device provided with a collagen gel on which the tissue equivalent is arranged, the tissue equivalent forming the first region and the permeable Element forms the second area. 12. Device for determining the interaction of tissue and at least one agent using at least one tissue equivalent, which device comprises the tissue equivalent and at least one container for the tissue equivalent, wherein the container comprises: (i) an inner container comprising a permeable means provided with a collagen gel on which the tissue equivalent is disposed, the permeable means forming the bottom of the inner container, and (ii) an outer container within which the inner container is arranged. 13. The apparatus of claim 12, wherein the apparatus further comprises a means for closing the container, which is removably sealable to the container. 14. Device according to claim 12, wherein the inner container is provided with at least one opening which communicates with the outer container. 15. The device of claim 12, wherein the inner container contains K-resin. 16. The device of claim 12, wherein an absorbent element is located beneath the permeable element. 17. The device of claim 14, wherein the absorbent element comprises a fiber pad or a gel. 18. The device of claim 17, wherein the fiber pad is cotton. 19. The device of claim 12, wherein an agarose-containing gel is disposed in the outer container.